The invention relates to a method of preparation of facial metal tricarbonyl compounds and further co-ordinated facial metal tricarbonyl compounds. The invention further relates to the use of said facial metal tricarbonyl compounds in the labelling of biologically active substrates and other ligands, and to a kit for preparing a facial metal tricarbonyl compound or further co-ordinated facial metal tricarbonyl compounds.
The application of metal complexes, with a wide variety of radionuclides, in the field of nuclear medicine has become a major tool in diagnosis and also more recently in therapy. The metal complexes are often attached to a biologically active substrate that acts as a targeting agent. One of the most widely applied procedures for the metal-labelling of biologically active substrates such as proteins, peptides, sugars or small biologically active compounds consists in stabilizing the M(V)=O moiety of (radioactive) metals of group 7B of the periodic table with different tetradentate ligands. After reduction, the M(V)=O moiety is intermediately stabilized with a larger amount of an auxiliary ligand such as glucoheptonate which is subsequently substituted by the chelator attached to the system to be labelled. This method has proven to be successful in many cases but suffers from some major disadvantages such as the required high denticity and the bulkiness of the ligand and the difficulty in synthesizing and attaching such ligand.
It is known in the art (Alberto et al., J. Nucl. Biol. and Med. 1994, 38, 388-90) that facial metal tricarbonyl complexes of radioactive metals of group 7B of the periodic table are very convenient starting materials for substitution reactions in organic solvents as well as in water, as these compounds are stable in water for weeks, even if exposed to air. Therefore said compounds would be very useful for the labelling of biologically active substrates, such as amino acids, peptides, proteins, sugars and any receptor binding molecules. A major drawback, however, of these compounds until now is that they have only been available from high temperature carbonylation reactions and with the aid of the pyrophoric and toxic and therefore dangerous reducing agent BH3 (Alberto et al., Low CO pressure synthesis of (NEt)2[MX3(CO)3] (M=Tc, Re) and its Substitution Behaviour in Water and Organic Solvents. Technetium in Chemistry and Nuclear Medicine, No 4, Cortina International, Milano, 1994).
It is the objective of the present invention to provide for a method of preparing facial metal tricarbonyl compounds of (radioactive) metals of group 7B with the aid of easily available and low-toxic starting materials at moderate temperature and at normal pressure of CO, in a reasonable time and with high yield. Such a method would be a powerful tool that can be used for the synthesis of diagnostic and therapeutic agents, especially for the synthesis of said diagnostic and therapeutic agents derived from radioactive metals with a short lifetime, in order to have access to these labelled compounds in poorly-equipped hospital laboratories. When the above mentioned diagnostic agent is labelled with a radionuclide it can be detected by the so-called single photon emission computerized tomography (SPECT and SPET), when it is labelled with a paramagnetic metal atom it can be detected by magnetic resonance imaging.
The above-defined objective can be achieved, according to the present invention, by a method of preparing a compound of the general formula
fac-[M(CO)3(OH2)3]+xe2x80x83xe2x80x83(I)
wherein M is Mn, 99mTc, 186Re or 188Re,
by reacting a metal in the permetallate form (MO4xe2x88x92 form) with carbon monoxide and a reducing agent, characterized in that a mixture of a base, a reducing agent soluble in water but not substantially decomposed by water, and optionally a stabilizing agent is solved in a water containing solvent system containing a solution of the metal in the permanganate, pertechnetate or perrhenate form in the presence of carbon monoxide. The metal M is preferably 99mTc, 186Re or 188Re, as these radionuclides, when used in diagnostic or therapeutic agents, have the advantage that they can be applied in very low concentrations, which minimizes the risk of toxicity.
The term xe2x80x9cnot substantially decomposed by waterxe2x80x9d means that upon the addition of the solution of permanganate, pertechnetate or perrhenate in water, the velocity of the decomposition reaction of the reducing agent with water is zero or very low compared with the reaction of said reducing agent with the permanganate, pertechnetate or perrhenate, so that the reaction with said permetallate is completed when still enough of the reducing agent is present. It is very surprising that a quantitative reduction of permetallates in water containing solvent systems can be achieved at moderate temperature and in reasonable times with reducing agents that are nucleophilic and that are generally considered as less reactive than the electrophilic reducing agent BH3 known in the art.
The method of the invention can be easily performed just by mixing the permetallate solution with the other reagents in the presence of carbon monoxide. The permetallate solution may optionally contain halide ions needed for the elution of the permetallate from a generator. The carbon monoxide may be supplied by using a closed system with an atmosphere containing a sufficient amount of carbon monoxide, or by flushing the carbon monoxide gas through the solution. The base used is preferably an inorganic base, selected from the group of stable hydroxides and carbonate salts such as NaOH, KOH, NaHCO3, Na2CO3, KHCO3, K2CO3, Ca(OH)2 and Mg(OH)2. Most preferred is Na2CO3. The base is added in a molar ratio to the reducing agent of between 0.1 and 2, and preferably in a molar ratio of approx. 0.35. The reaction can be performed with and without a stabilizing agent. As a stabilizing agent gentisate (2,5-dihydroxybenzoate), glucoheptonate, citrate or tartrate can be used. The preferred stabilizing agent is tartrate, e.g. as NaK-tartrate. The stabilizing agent is added to the reaction mixture in such an amount that its concentration is higher than that of the metal to be reduced. For the reduction several reducing agents can be used, such as borohydride anion (BH4xe2x88x92) or substituted borohydride anion wherein up to three of the hydrogen atoms which comprise the borohydride anion have been independently replaced by inert substituents. Examples of said inert substituents are alkoxy or alkylcarbonyloxy groups containing 1 to 10 carbon atoms and cyano groups. The counterion of the reducing group may consist of a metal of group 1A or 2A of the periodic table or zinc or an ammonium or tetrasubstituted ammonium or tetrasubstituted phosphonium ion, wherein the four substituents are each independently alkyl groups containing from 1 to 10 carbon atoms, hydroxyalkyl groups or alkoxyalkyl groups containing from 2 to 10 carbon atoms or aryl groups.
Preferred reduction reagent is borohydride anion, especially in the form of compounds such as sodium borohydride, potassium borohydride, lithium borohydride and zinc borohydride. Most preferred reducing agent is sodium borohydride. The reducing agent is reacted with the permetallate in a molar ratio higher than 3. The reduction reaction can be performed at a temperature between 20xc2x0 C. and 100xc2x0 C. The preferred reaction temperature is approx. 75xc2x0 C. The heating of the reaction mixture can be performed in the normal way but also by micro-wave heating. The reaction can also be performed by the application of ultra sound, e.g. by carrying out the reactions in an ultrasonic bath at room temperature, normally leading to the same reaction rate at lower reaction temperature.
The compound of the general formula (I) obtained is very suitable for the labeling of biologically active substrates, such as amino acids, peptides, proteins, sugars, small receptor binding molecules or cells. Examples of peptides that may be labelled are growth factors, somatostatin, bombesin, insulin, LHRH, gastrin, gastrin releasing peptide, thyrotropin releasing hormone, thyroid stimulating hormone, prolactin, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), angiotensin, neurotensin, interferons, IL-1, IL-4 and IL-6, monoclonal antibodies and their analogues and derivatives. After labelling with a suitable labelling substance these peptides can e.g. be used in the detection and localisation or treatment of malignant human tumours.
Examples of sugars that may be labelled are glucose and deoxyglucose and derivatives of said compounds.
Small receptor binding molecules are defined as non-peptide molecules which are binding to a receptor and normally have a molecular mass below approximately 500 Daltons. Examples of small receptor binding molecules that may be labelled are substances for the serotonergic system as described in WO 96/30054, or substances for the dopaminergic system (e.g. raclopride, xcex2-CIT, lisuride), for the cholinergic system (e.g. epibatidine), for the glutaminergic system (e.g. mematine) or for the benzodiazepine system (e.g. flumazenil, iomazenil). Examples of metabolic active molecules that may be labelled are DOPA, Tyrosine, mIBG, MAO-I and analogues thereof.
Examples of cells that may be labelled are red and white blood cells.
As a result of the labeling of (biologically active) substrates with a compound of the general formula I, a further coordinated compound of the general formula
fac-[M(CO)3(X)2L1]nxe2x80x83xe2x80x83(II),
fac-[M(CO)3(X)L2]nxe2x80x83xe2x80x83(III)
or
fac-[M(CO)3L3]nxe2x80x83xe2x80x83(IV),
wherein:
M is Mn, 99mTc, 186Re or 188Re;
L1 is a monodentate ligand,
L2 is selected from the group consisting of a bidentate ligand and two monodentate ligands, and
L3 is selected from the group consisting of a tridentate ligand, a monodentate ligand and a bidentate ligand, and three monodentate ligands;
X is H2O or a halide ion;
n the sum of the charge of the ligands L1 or L2 or L3 and X increased with one + charge
is obtained.
After the labeling reaction the ligand X is usually H2O. One of the H2O ligands may, however, be replaced by a halide ion, when available, to neutralize the charge of the complex. This is often the case for compounds of the general formula Ill. When the ligand L1, L2 or L3 before and/or after labeling with the facial metal tricarbonyl compound is the biologically active molecule, the present invention gives easy access to compounds that directly can be used as a diagnostic and therapeutic agent.
Examples of monodentate ligands within the definition of L1, L2 and L3 are (biologically active) substrates bearing groups such as phosphines, isonitriles, nitrites, imidazoles, thioethers and pyridine-like aromatic amines. Examples of bidentate ligands within the definition of L2 and L3 are (biologically active) substrates bearing pyridin, imidazole or pyrazole groups, such as histidine, histamine, functionalized imidazole systems, bidentate thioethers, bidentate isocyanides, Schiff-base type ligands and picolinic acid.
Examples of tridentate ligands within the definition of L3 are tris-pyrazolyl borate, tris-pyrazolylmethane, tris-imidazolyl borate, tris-pyrazolylmethane, 1,4,7-trithiacyclononane (9-aneS3) and triazacyclononane (9-aneN3), histidine, methionine, cystein derivatized at the thiol group to give a thioether and cyclopentadienyl derivatives.
In some cases it may be advantageous to prepare the radiolabelled bioactive compound in one step. This objective can be achieved according to the present invention, with a method of preparing a compound of the general formula
fac-[M(CO)3(X)2L1]nxe2x80x83xe2x80x83(II),
fac-[M(CO)3(X)L2]nxe2x80x83xe2x80x83(III)
or
fac-[M(CO)3L3]nxe2x80x83xe2x80x83(IV),
wherein:
M is Mn, 99mTc 186Re or 188Re;
L1 is a monodentate ligand,
L2 is selected from the group consisting of a bidentate ligand and two monodentate ligands, and
L3 is selected from the group consisting of a tridentate ligand, a monodentate ligand and a bidentate ligand, and three monodentate ligands;
X is H2O or a halide ion;
n the sum of the charge of the ligands L1 or L2 or L3 and X increased with one + charge;
characterized in that a mixture of a base, ligands L1 or L2 or L3, a reducing agent soluble in water but not substantially decomposed by water, and optionally a stabilizing agent is solved in a water containing solvent system containing a solution of the metal in the permanganate, pertechnetate or perrhenate form in the presence of carbon monoxide and optionally in the presence of halide.
Especially in the case of radiolabelled compounds it is frequently impossible to put the ready-for-use composition at the disposal of the user, in connection with the often poor shelf life of the radiolabelled compound and/or the short half-life of the radionuclide used. In such cases the user will carry out the labelling reaction with the metal in the clinical hospital or laboratory. For this purpose the various reaction ingredients are then offered to the user in the form of a so-called xe2x80x9ckitxe2x80x9d. It will be obvious that the manipulations necessary to perform the desired reaction should be as simple as possible to enable the user to prepare from the kit the radioactive labelled composition by using the facilities that are at his disposal. Therefore the invention also relates to a kit for preparing a labelling composition, which labelling composition contains compound of formula I as the labelling agent. Such a kit for the labelling of a biologically active substrate, according to the present invention, comprises (i) a reducing agent soluble in water but not substantially decomposed by water, (ii) a base, (iii) if desired, a stabilizing agent and/or a chelator and (iv) if desired one or more inert pharmaceutically acceptable carriers and/or formulating agents and/or adjuvants, at least one of said ingredients (i) to (iv) being stored in a container having an atmosphere containing a sufficient amount of carbon monoxide, said ingredients (i) to (iv) optionally independently being combined, and (v) instructions for use with a prescription for reacting the ingredients of the kit with a metal selected from the group consisting of Mn, 99mTc, 186Re or 188Re in the form of a permetallate solution.
It is the merit of the present invention, disclosing an easy way of preparing facial tricarbonyl metal compounds within a time-frame that is reasonable compared with the half-life time of the radioactive isotopes involved, and with high yields, that a kit can be prepared for the labelling of biologically active substrates with said facial tricarbonyl metal compounds.
In some cases it may be advantageous to enclose a bioactive substrate in the kit so that a kit is obtained for the preparation of a radiopharmaceutical composition.
Alternatively the biologically active compound is formed upon the reaction of the ligand with the facial metal tricarbonyl compound. Such a kit for the preparation of a diagnostic and therapeutic pharmaceutical composition, according to a different embodiment of the present invention, comprises (i) a suitable substrate to be labelled with a metal selected from the group consisting of Mn, 99mTc, 186Re or 188Re, (ii) a reducing agent soluble in water but not substantially decomposed by water, (iii) a base, (iv) if desired, a stabilizing agent and/or a chelator, (v) if desired one or more inert pharmaceutically acceptable carriers and/or formulating agents and/or adjuvants, at least one of said ingredients (i) to (v) being stored in a container having an atmosphere containing a sufficient amount of carbon monoxide, said ingredients (i) to (v) optionally independently being combined, and (vi) instructions for use with a prescription for reacting the ingredients of the kit with said metal in the form of a permetallate solution.
The preparation of the diagnostic and therapeutic pharmaceutical composition with the aid of the above mentioned kit enclosing a (biologically active) substrate can take place in two alternative embodiments. In the first embodiment the facial tricarbonyl metal compound is prepared first and then reacted with the substrate to be labelled. In the second embodiment the reduction step is carried out in the presence of the substrate to be labelled, directly leading to the labelled compound.